In all eukarotic cells, the endoplasmic reticulum (ER) forms a highly fenestrated network that spreads throughout the volume of the cell, often lying just below the plasma membrane. We have taken a genetic approach towards understanding the mechanism of ER dynamics, structure and inheritance in yeast. Our work to date shows that ER tubules form from the nuclear envelope at the start of the cell cycle, they are delivered into the bud along actin cables by the type V myosin, Myo4p, they are anchored at the bud tip by the exocyst complex and then spread along the cell cortex to form the cortical ER. We propose that Rtn1 p, a member of the conserved reticulon family of proteins, functions as the receptor for the exocyst on the ER membrane and that Rtnlp also serves to link the ER to the cell cortex. We further propose that ergosterol and sphingolipids play an important role controlling the mobility or activity of a key component of the ER inheritance machinery at the cell cortex. Five specific aims are planned: 1) We will analyze the interaction of Rtn1 p with the exocyst, defining the topology of Rtnlp within the ER membrane and the specific protein-protein interactions that link the exocyst to Rtnlp. 2) We will determine if Rtnlp binds to a protein along the cortex and if this interaction underlies the unique localization of Rtnlp as well as the tight association of the ER with the cell cortex. 3) We will determine if Rtnlp plays a role in forming the fenestrated reticulum characteristic of the ER in all eukaryotes and if it interacts with other components of the ER membrane in this capacity. 4) We will use the information from our analysis of Rtnlp in yeast to construct dominant negative alleles of mammalian reticulon proteins. Phenotypic analysis will determine if the roles of Rtn1 p are conserved. 5) We will determine if ergosterol and sphingolipids are critical for the localization, mobility or activity of a cortical component of the machinery that establishes the structure, dynamics and inheritance of the ER. [unreadable] [unreadable]